EP0218352A1 - Verfahren zur Behandlung von Protistezellen - Google Patents

Verfahren zur Behandlung von Protistezellen Download PDF

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Publication number
EP0218352A1
EP0218352A1 EP86306599A EP86306599A EP0218352A1 EP 0218352 A1 EP0218352 A1 EP 0218352A1 EP 86306599 A EP86306599 A EP 86306599A EP 86306599 A EP86306599 A EP 86306599A EP 0218352 A1 EP0218352 A1 EP 0218352A1
Authority
EP
European Patent Office
Prior art keywords
cells
protista
suspension
process according
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP86306599A
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English (en)
French (fr)
Other versions
EP0218352B1 (de
Inventor
Akitoshi Ito
Kazumasa Takahashi
Shozo Shiozaki
Shigeru Morikawa
Akira Katayama
Takeshi Udagawa
Takeshi Kobayashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zeon Corp
IHI Corp
Original Assignee
IHI Corp
Nippon Zeon Co Ltd
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Publication date
Application filed by IHI Corp, Nippon Zeon Co Ltd filed Critical IHI Corp
Publication of EP0218352A1 publication Critical patent/EP0218352A1/de
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/38—Nucleosides
    • C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves

Definitions

  • This invention relates to a process for treating protista cells having a useful substance accumulated therein, more particularly a process for recovering the useful substance in the cells by irradiating the cells with laser beam.
  • the useful substances include, for example, enzymes and proteins such as trypsin, amylase, cellulase, lysozyme, ribonuclease, cytochrome, insulin, pepsin and single cell proteins (SCP); amino acids such as lysine, glutamic acid, leucine, tryptophan, arginine, homoserine and methionine; nucleic acids such as nucleosides, nucleotides, oligonucleotides, DNA and RNA; vitamins such as vitamin B 1 , vitamin B 2' vitamin B 6 and vitamin H; and biologically active substances such as S-adenosyl-L-methionine (hereinafter referred to as SAM in some cases), S-adenosylhomocysteine, glutathione, interferon and interleukin.
  • enzymes and proteins such as trypsin, amylase, cellulase, lysozyme, ribonuclea
  • the mechanical process and the ultra- sonication process are disadvantageous in that since destruction of cell walls results in discharge of all substances in cells, a very troublesome procedure is required for thereafter selectively separating and purifying a useful substance, for example, only a protein having a molecular weight in a specific range.
  • the process comprising bacteriolysis by autolysis and the process comprising treatment with a cell wall-degrading enzyme are applicable only to limited kinds of protista.
  • the treatment with an agent such as perchloric acid permits substantially complete extraction of a useful substance in cells but has been disadvantageous in that it requires a subsequent neutralization step or results in decomposition of the useful substance or toxicity.
  • the present inventors have devoted themselves to research on a process for treating cells which can solve the above-mentioned problems in the conventional treatments of cells, comprises simpler steps, and is applicable to various kinds of protista, and have consequently found that a useful substance can easily be discharged from cells by irradiation of the cells with laser beam, whereby this invention has been accomplished.
  • the object of this invention is to provide a process for treating protista cells which makes it possible to extract a useful substance from a variety of protista cells by a simple procedure.
  • Such a process of this invention can be achieved by irradiating protista cells having a useful substance therein with laser beam to discharge the useful substance from the cells.
  • Fig. 1 and Fig. 2 show analysis charts of HPLC of the proteins obtained in Example 1 and Example 6, respectively.
  • protista in a wide range of from higher protista to lower protista.
  • the protista include bacteria such as Escherichia, Pseudomonas, Bacillus, Brevibacterium, Corynebacterium, Serratia, Citrobacter, Streptococcus, Lactobacillus, Staphylococcus and the like; fungi such as Aspergillus, Penicillium, Mucor, Rhizopus, Gibberella, Monascus, Neurospora, Botrytis, Cladospolium, Fusarium and the like; yeasts such as Saccharomyces, Candida, Pichia, Hansenula, Schizosaccharomyces, Lipomyces, Rhodotolula and the like; Actinomycetes such as Streptomyces, Nocardia, Actinomyces and the like; Bacidiomycetes such as Lentinus, Trametes, Coprinus, Pycn
  • the laser beam used in this invention usually has a specific wavelength in the range of from the ultraviolet region to the infrared region, and its output mode may be either CW (continuous wave) or pulse.
  • the laser beam is preferably pulsed. In this case, a rise of temperature due to light absorption of the substance can be prevented by a non-irradiation time between pulses, so that even a useful substance unstable to heat becomes extractable in a stable state.
  • the laser beam used includes solid-state lasers such as YAG laser, glass laser, ruby laser and the like, a dye laser, and gas lasers such as excimer laser, argon ion laser and the like. Furthermore, lasers pumped by other laser such as YAG laser pumped by dye laser, excima laser pumped by dye laser, argon ion laser pumped by dye laser and the like may be used.
  • the treatment of cells with laser beam is usually carried out by suspending the cells in a vehicle, for example, water, a buffer solution or an organic solvent such as an alcohol, ketone, ester, ether, hydrocarbon or the like, and irradiating the resulting suspension with laser beam.
  • a vehicle for example, water, a buffer solution or an organic solvent such as an alcohol, ketone, ester, ether, hydrocarbon or the like, and irradiating the resulting suspension with laser beam.
  • the mode of the treatment with.laser beam may properly be selected depending on purposes, and any of batch mode, semicontinuous mode and continuous mode can be employed.
  • the suspension of protista cells are irradiated with laser beam.
  • the treatment is carried out by feeding the suspension of protista cells to a laser beam irradiation zone intermittently or continuously.
  • a method for feeding the aforesaid suspension is not critical and includes, for instance, a method comprising feeding the suspension from the upper part of the laser beam irradiation zone and taking out the same from the lower part; a method comprising feeding the suspension from the lower part and taking off the same from the upper part; and a method comprising passing the suspension through the laser beam irradiation zone transversely.
  • the semicontinuous mode or the continuous mode is preferred among these treatment modes, and according to them, the efficiency of treatment can be raised by recycling the cell suspension after the treatment to the laser beam irradiation zone.
  • the recycling it is also possible to separate cells by a conventional method such as centrifugation, then prepare a suspension thereof again and feed the same.
  • the content of cells in the cell suspension is usually 0.01 to 50% by weight, preferably 0.1 to 20% by weight in terms of the weight of wet cell.
  • the temperature of the cell suspension is not critical so long as it causes no denaturation of a desired useful substance, it is usually 0° to 100°C, preferably 0° to 50°C.
  • the irradiation time of laser beam is usually tens of seconds to about 10 hours, preferably 1 minute to 2 hours.
  • the irradiation dose of laser beam is varied depending on the kind of cells, the kind of a substance to be extracted, and the like, it can easily be determined by carrying out a simple preliminary experiment.
  • the recovery method includes, for example, a method comprising directly obtaining the useful substance by extraction with a solvent, and a method comprising removing a cell debris by cetrifugation or the like and recovering the useful substance from the thus obtained supernatant by a technique such as ion-exchange chromatography, gel filtration, electrophoresis, solvent precipitation, salting-out or the like.
  • intracellular useful substances can be extracted from various kinds of protista cells very efficiently by a simple procedure.
  • Saccharomyces cerevisiae IFO-2044 was cultured in the medium of Schlenk, F. et al. [see J. Biol. Chem. 229, 1037 (1957)] and 1 g (wet weight) of the S-adenosylmethionine (SAM)-containing cells thus obtained were suspended in 99 ml of distilled water. Then, 3ml of the resulting suspension was placed in a quartz cuvette and irradiated with a YAG laser pumped by dye laser system (wavelength 266 nm, l5mJ/pulse, 10Hz) for 10 minutes with stirring by means of a stirrer. The irradiated suspension was centrifuged to remove a cell debris, and the amount of SAM in the supernatant thus obtained was measured by HPLC to be 42.9 mg.
  • SAM S-adenosylmethionine
  • the protein concentration in the supernatant obtained by the procedure described above was measured to be 0.46 mg.
  • a suspension of Igof wet cells in 99 ml of 0.2M patassium phosphate buffer (pH 7.5) containing 5mM dithiothreitol (hereinafter abbreviated as DTT) was ground and stirred by means of a DYNO-MILL (mfd. by Willy. A. Bachofen A.G.) at 4,500 r.p.m. for 11 minutes by using glass beads (0.25 to 0.5 mm), to disrupt the cells.
  • the suspension was centrifuged to remove a cell debris and the glass beads, and the protein concentration in the cell-free extract thus obtained was measured to be 0.97 mg. That is to say, protein was extracted by the laser irradiation in an amount of 47% of the amount of protein extracted by means of the DYNO-MILL.
  • Escherichia coli ATCC-14948 was inoculated into a liquid medium consisting of lg/dl of glucose, 1.5g/dl of peptone, 0.3g/dl of yeast extract, 0.3g/dl of K 2 HP0 4 , 0.2g/dl of NaCl and 0.02g/dl of MgSO 4 ⁇ 7H 2 O which had been adjusted to pH 7.0 and sterilized by heating, and it was subjected to shaking culture at 28°C for 16 hours.
  • the culture broth was centrifuged and 1 g (wet weight) of the cells thus obtained were suspended in 99 ml of 0.02M potassium phosphate buffer (pH 7.5) containing 5mM DTT.
  • the percentage of extraction of protein by the laser irradiation was calculated to be 98.7% based on the protein extracted by the ultrasonic cell disintegration.
  • Penicillium oxalicam IFO-5748 was inoculated into a liquid medium consisting of 5g/dl of glucose, 0.5g/dl of peptone, O.lg/dl of yeast extract, 2.0g/dl of KH 2 PO 4 , O.lg/dl of K 2 HPO 4 and 0.02g/dl of MgSO 4 ⁇ 7H 2 O which had been adjusted to pH 6.5 and sterilized by heating, and it was subjected to shaking culture at 28°C for 48 hours. The culture broth was centrifuged and 1 g (wet weight) of the cells thus obtained were suspended in 99 ml of 0.02M potassium phasphate buffer (pH 7.5) containing 5mM DTT.
  • 0.02M potassium phasphate buffer pH 7.5
  • Streptomyces hygroscopicus IFO-3192 was inoculated into a liquid medium consisting of lg/dl of peptone, 0.5g/dl of meat extract, O.lg/dl of yeast extract and 0.5g/dl of NaCA which had been adjusted to pH 7.0 and sterilized by heating, and it was subjected to shaking culture at 34°C for 72 hours.
  • the culture broth was centrifuged and 1 g (wet weight) of the cells thus obtained were suspended in 99 ml of 0.02M potassium phosphate buffer (pH 7.5) containing 5mM DTT.
  • the percentage of extraction of protein by laser irradiation was calculated to be 90.3% based on the protein extracted by the ultrasonic cell disintegration.
  • the extraction percentage in the case of this process was calculated to be 70.7% from the fact that in Example 1, the SAM content of the cells was 55.0 mg.
  • Pycnoporus coccineus IFO 6489 was inoculated into a liquid medium consisting of 5g/dl of glucose, 0.5g/dl of peptone, O.lg/dl of yeast extract, 0.2g/dl of KH 2 P0 4 , O.lg/dl of K 2 HPO 4 and 0.02g/dl of MgSO 4 ⁇ 7H 2 O which had been adjusted to pH 6.5 and sterilized by heating, and it was subjected to shaking culture at 28°C for 72 hours. The culture broth was filtered and 1 g (wet weight) of the cells thus obtained were suspended in 99 ml of 0.02M potassium phosphate buffer (pH 7.5) containing 5mM DTT.
  • 1g (wet weight) of the same cells as described above were suspended in 99 ml of 0.02M potassium phosphate buffer containing 5mM DTT, and the resulting suspension was treated by means of a DYNO-MILL (mfd. by Willy. A. Bachofen A.G.) at 4°C at 4,500 r.p.m. for 11 minutes by using glass beads for DYNO-MILL (0.25 to 0.5 mm), to grind the cells.
  • the suspension treated was centrifuged to remove cell debris and the glass beads, and the protein concentration in the cell-free extract thus obtained was measured to be 9.8 mg/g wet cell. From this result, the percentage of extraction of protein by laser irradiation was calculated to be 87.7% based on the protein extracted by the grinding by means of the DYNO-MILL.
EP19860306599 1985-08-29 1986-08-27 Verfahren zur Behandlung von Protistezellen Expired EP0218352B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP19061285A JPS6251990A (ja) 1985-08-29 1985-08-29 微生物菌体の処理方法
JP190612/85 1985-08-29

Publications (2)

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EP0218352A1 true EP0218352A1 (de) 1987-04-15
EP0218352B1 EP0218352B1 (de) 1991-07-10

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EP (1) EP0218352B1 (de)
JP (1) JPS6251990A (de)
DE (1) DE3680172D1 (de)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001630A1 (en) * 1987-08-12 1989-02-23 Nemeth Peter Procedure for relieving cell mixtures and tissues of unwanted populations
AT389889B (de) * 1988-05-03 1990-02-12 Langenecker Bertwin Dr Verfahren und vorrichtung zum inaktivieren von in einem traegermedium enthaltenen viren
FR2815640A1 (fr) * 2000-10-19 2002-04-26 Centre Nat Rech Scient Procede de production de proteines par electropulsation de levures
US7282002B2 (en) * 2003-07-04 2007-10-16 Honda Motor Co., Ltd. Belt for continuously variable transmission

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4292408A (en) * 1975-12-24 1981-09-29 Kernforschungsanlage Julich Gesellschaft Mit Beschrankter Haftung Mass and a method of preparing same of living cells of organisms for adsorbing metal ions from a physiological solution, and employment of the mass for enriching metals
EP0137504A2 (de) * 1983-10-13 1985-04-17 Rikagaku Kenkyusho Verfahren und Apparat zum Einpflanzen eines Fremdstoffes in lebende Zelle

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4292408A (en) * 1975-12-24 1981-09-29 Kernforschungsanlage Julich Gesellschaft Mit Beschrankter Haftung Mass and a method of preparing same of living cells of organisms for adsorbing metal ions from a physiological solution, and employment of the mass for enriching metals
EP0137504A2 (de) * 1983-10-13 1985-04-17 Rikagaku Kenkyusho Verfahren und Apparat zum Einpflanzen eines Fremdstoffes in lebende Zelle

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 104, no. 5, February 1986, page 298, abstract no. 31399g, Columbus, Ohio, US; & JP-A-60 164 487 (HITACHI LTD.) 27-08-1985 *
SCIENCE, vol. 213, 31st July 1981, pages 505-513, AAAS; M.W. BERNS et al.: "Laser microsurgery in cell and developmental biology" *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989001630A1 (en) * 1987-08-12 1989-02-23 Nemeth Peter Procedure for relieving cell mixtures and tissues of unwanted populations
AT389889B (de) * 1988-05-03 1990-02-12 Langenecker Bertwin Dr Verfahren und vorrichtung zum inaktivieren von in einem traegermedium enthaltenen viren
FR2815640A1 (fr) * 2000-10-19 2002-04-26 Centre Nat Rech Scient Procede de production de proteines par electropulsation de levures
WO2002033065A3 (fr) * 2000-10-19 2003-05-30 Centre Nat Rech Scient Procede de production de proteines par electropulsation
US7282002B2 (en) * 2003-07-04 2007-10-16 Honda Motor Co., Ltd. Belt for continuously variable transmission

Also Published As

Publication number Publication date
EP0218352B1 (de) 1991-07-10
DE3680172D1 (de) 1991-08-14
JPS6251990A (ja) 1987-03-06

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